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Recommendations for Successful siRNA Library Screens

ontrol transfected cells. Interestingly, the hits identified by the pools of siRNAs only partially overlapped with the hits identified by the three individually transfected siRNAs (Figure 3A).

Since our definition of a validated hit is a gene for which two siRNAs yield the same phenotype in a screen, we were able to use the results of the screen to determine if the pool results could be validated with the individual siRNA results. Indeed, only 60% of the siRNA pool hits could be validated with the results of the individual siRNAs. Non-validated hits were considered false positives. The pools gave a false positive rate of 40%.

Of greater concern was that six of the twelve genes for which two or more single siRNAs yielded a hit, failed to register as hits in the siRNA pool screen. The 50% false negative rate observed for pools indicates that screening with siRNA pools can result in lost opportunities for target gene identification. A sampling of confirmed positives, false positives, false negatives, and confirmed negatives is shown in Figure 3B.

We have measured the false positive and false negative rates associated with screening using a single siRNA for each gene target or pools of three siRNAs. The siRNA pool versus individual siRNA screen analysis was repeated using nine different phenotypic assays including cell proliferation, cell morphology, and cytoskeleton formation assays. The pools consistently produced false positive rates of greater than 50% and false nega
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